BMP4 promotes hepatocellular carcinoma proliferation by autophagy activation through JNK1-mediated Bcl-2 phosphorylation

Abstract

Background: Autophagy is a conserved catabolic process with complicated roles in tumor development. Bone morphogenetic protein 4 (BMP4), a member of the transforming growth factor (TGF-β) family of regulatory proteins, plays a crucial role in human malignancies. However, whether BMP4 contributes to the regulation of autophagy in hepatocellular carcinoma (HCC) progression remains elusive.

Methods: Functional analysis of BMP4 on HCC proliferation and autophagy was performed both in vitro and in vivo in HepG2 and HCCLM3 cells. Autophagic activity was estimated by Western blot for autophagic marker proteins and by transmission electron microscopy (TEM). Transfection of mRFP-GFP-LC3 adenovirus was applied to observe autophagic flux and high content screening was used for quantification. The signaling pathway of BMP4-regulated HCC proliferation and autophagy was investigated by Western blot.

Results: BMP4 treatment promoted HCC cells proliferation and induced autophagy. The in vivo xenograft model supported that BMP4 overexpression promoted the growth of HCC cells and autophagy induction while BMP4 knockdown exerted the opposite effect. 3-MA pre-treatment or knockdown of Beclin-1 (BECN1) blocked HCC autophagy by decreasing the expression of LC3-II and subsequently attenuated BMP4-induced autophagy and cells proliferation enhanced by BMP4 in vitro and in vivo. Mechanistic study revealed that the induction of autophagy by BMP4 was mediated through activating the JNK1/Bcl2 pathway. Furthermore, the JNK1 inhibitor and knockdown of JNK1 could attenuate autophagy induced by BMP4 and eliminated BMP4-promoted HCC cells growth.

Conclusions: BMP4 promoted HCC proliferation by autophagy activation through JNK1/Bcl-2 signaling.

Keywords: Autophagy; BMP4; Hepatocellular carcinoma; JNK1; Proliferation.

Fig. 1

BMP4 induced autophagy in HCC cell lines: a & b HepG2 and HCCLM3 cell lines were treated with 100 ng/mL BMP4 recombinant protein for different time points (0 h, 1 h, 6 h, 12 h, 24 h and 48 h) to evaluate the effects on autophagy. Western blot was applied to detect the expression levels of LC3-II, p62 and BECN1. The expression levels of LC3-II were quantified by Image lab software by densitometric analysis and were normalized to the control groups. GAPDH was used as the internal control. n = 3, one-way ANOVA with post-hoc Tukey’s test. c & d Representative images of intracellular double-membrane vesicles (red arrows), the ultrastructural feature of autophagy, detected by TEM in HCC cells. HepG2 and HCCLM3 cells were treated with 100 ng/mL BMP4 or 200 ng/mL Noggin for 24 h

Fig. 2

BMP4 activates autophagic flux in HCC cell lines: a HCC cell lines were pre-treated with autophagy inhibitor 3-MA for 1 h before BMP4 administration to prevent autophagy activated by BMP4. Western blot was applied to detect the expression levels of LC3-II, p62 and BECN1 in both HepG2 and HCCLM3 cells. b GAPDH was used as the internal control. Quantification analysis of LC3-II / GAPDH in Fig. 3 (a) was normalized to the control (blank) groups. n = 3, one-way ANOVA with post-hoc Tukey’s test. c Representative fluorescent images of mRFP-GFP-LC3 transfection. mRFP-GFP-LC3-HepG2 / HCCLM3 cells were exposed to BMP4, BMP4 with 3-MA and Noggin for 24 h. The fluorescent images were obtained from the Operetta automated microscope (Original magnification: × 200). The yellow puncta indicated autophagosomes and red puncta represent autolysosomes. d Quantification autophagic flux was calculated by red puncta in the merged images with Perkin-Elmer Columbus 2.3 software. n = 3, one-way ANOVA with post-hoc Tukey’s test

Fig. 3

BMP4 activated-autophagy promoted HCC cells proliferation in vitro: a & b HepG2 and HCCLM3 were treated with autophagy inhibitor 3-MA, BMP4 + 3-MA and BMP4 alone. CCK-8 assays were applied to detect cell viability in HCC cells with different treatment. n = 3, one-way ANOVA with post-hoc Tukey’s test. c & d Effects of autophagy inhibitor 3-MA on BMP4 promoted-colony formation ability in HCC cells. Combination of 3-MA and BMP4 significantly decreased the number of colonies as compared to the BMP4-treated groups. n = 3, one-way ANOVA with post-hoc Tukey’s test. e Transfection efficiency of siRNA targeting BECN1 in HCC cells was confirmed by Western blot. f The expression level of LC3-II was detected by Western blot. Knockdown of BECN1 decreased the expression of LC3-II as compared with the control groups and prevented autophagy activated by BMP4. g & h Knockdown of BECN1 significantly attenuated cell viability promoted by BMP4 both in HepG2 and HCCLM3 cells. Cell viability was determined by CCK-8. n = 3, one-way ANOVA with post-hoc Tukey’s test

Fig. 4

BMP4 accelerated HCC cells growth by autophagy induction in vivo: a upper: Tumor formation of LV-BMP4 and LV-NC treated with 3-MA in HepG2 cells; lower: Tumor formation of si-BMP4 and si-NC treated with Rapa in HCCLM3 cells. b The average tumor weight of BMP4 overexpression in HepG2 cells was significantly more than the control group, while knockdown of BMP4 in HCCLM3 cells decreased the tumor weight. Autophagy inhibition by 3-MA attenuated the growth-promotion effect of BMP4 overexpression while autophagy induction restored the growth-suppression effect of BMP4 knockdown. n = 5, one-way ANOVA with post-hoc Tukey’s test. c The average tumor volume of subcutaneous implantation models of ectopic BMP4 expression and autophagy manipulation in HCC cells. n = 5, one-way ANOVA with post-hoc Tukey’s test. d HE staining and immunohistochemistry staining for the expression of BMP4, LC3B and proliferation-related protein Ki-67 in subcutaneous tumor (Original magnification: × 200)

Fig. 5

Signaling pathways involved in BMP4-activated autophagy to promote HCC proliferation: a Western blot was applied to detect the expression of JNK1, p-JNK, Bcl-2 and p-Bcl-2 in HepG2 and HCCLM3 cells. HCC cells were appointed to the indicated treatment respectively. b The effect of JNK inhibitor SP600125 was determined by Western blot in HepG2 and HCCLM3 cells. c Western blot was performed to detect the expression of LC3-II, p62 and BECN1 in HCC cells. JNK pathway inhibition significantly attenuated the BMP4-activated autophagy in HepG2 and HCCLM3 cells. d Quantification of LC3-II expression level by densitometric analysis and was normalized to the control (blank) groups. GAPDH was used as the internal control. n = 3, one-way ANOVA with post-hoc Tukey’s test. e Effects of JNK inhibitor SP600125 on long term colony formation promoted by BMP4 in HCC cells. JNK pathway inhibition significantly attenuated the promotion effects on the number of colonies both in HepG2 and HCCLM3. n = 3, one-way ANOVA with post-hoc Tukey’s test. f Effects of JNK inhibitor SP600125 on BMP4-promoted HCC cells growth. JNK pathway inhibition significantly attenuated the promotion effects on the cell viability both in HepG2 and HCCLM3

Fig. 6

Knockdown of JNK1 attenuated BMP4-activated autophagy and HCC proliferation: a Transfection efficiency of siRNA targeting JNK1 in HCC cells was confirmed by Western blot. b The expression level of LC3-II, p62 and BECN1 were detected by Western blot. Knockdown of JNK1 decreased BMP4-promoted LC3-II conversion as compared with the BMP4-treated group. c The expression levels of LC3-II were quantified by Image lab software by densitometric analysis and were normalized to the control groups. GAPDH was used as the internal control. n = 3, one-way ANOVA with post-hoc Tukey’s test. d Transfected cells were treated with BMP4 for 24 h. Cell viability was determined by CCK-8. n = 3, one-way ANOVA with post-hoc Tukey’s test